Cardiovascular disease remains the leading cause of morbidity and mortality in the United States; approximately 2600 deaths each day are the result of cardiovascular disease. In the United States, 50-60% of heart attacks occur in people without documented cardiovascular disease. A chief contributor to the pathology of the disease is the formation of atherosclerotic or “atheromatous” plaques in the coronary arteries (Farb et al. (1995) Circulation 92:1701-1709). An atheromatous plaque refers to a wide range of coronary lesions, from subtle collections of lipid, to obstructive coronary lesions that cause angina.
Atheromatous plaques can be active, and prone to rupture, or inactive and relatively stable. The progression of coronary atherosclerotic disease can be divided into five phases (Fuster et al. (1992) N Engl J Med 326:242-250). Phase I is represented by a small plaque that is present in most people under the age of 30 years, regardless of their country of origin, and that usually progresses slowly (i.e., type I-III lesions). Phase 2 is represented by a plaque, not necessarily very stenotic, with a high lipid content that is prone to rupture (i.e., type IV and Va lesions). The plaque of phase 3 may have predisposition to change in its geometry and to formation of mural thrombus, these processes by definition represent phase 3 (i.e., type I lesion), with a subsequent increase in stenosis, possibly resulting in angina, or ischemic sudden death. The mural and occlusive thrombi from plaques of phases 3 and 4, being organized by connective tissue, may contribute to the progression of the atherosclerotic process represented by severely stenotic or occlusive plaques of phase 5 (i.e., types Vb and Vc lesions). The severely stenotic plaques of phase 5, by a phenomenon of stasis and/or de-endothelialization, can become complicated by a thrombus and/or rapid myoproliferative response, also leading to an occlusive plaque of phase 5. Of interest is that about two thirds of coronary occlusions are the result of this late stenotic-type of plaque and are unrelated to plaque disruption. Unlike the rupture of less-stenotic lipid-rich plaques, leading to occlusion and subsequent infarction or other acute coronary syndromes, this process of occlusion from late stenotic plaques tends to be silent because the preceding severe stenosis and ischemia enhance protective collateral circulation (Fuster et al., (1992) N Engl J Med 326:242-250; Chesebro et al. (1992) Circulation 86 (suppl. 111)).
In general, atheromatous plaques characteristically comprise a fibrous cap surrounding a central core of extracellular lipids and debris located in the central portion of the thickened vessel intima, which is known as the “atheroma.” On the luminal side of the lipid core, the fibrous cap is comprised mainly of connective tissues, typically a dense, fibrous, extracellular matrix made up of collagens, elastins, proteoglycans and other extracellular matrix materials.
At the edges of the fibrous cap overlying an active atheromatous plaque, the lipid core comprises the shoulder region and is enriched with macrophages. The macrophages continually phagocytose oxidized LDL through scavenger receptors, which have a high ligand specificity for oxidized LDL. Continuous phagocytosis results in the formation of foam cells, a hallmark of the atherosclerotic plaque (Parthasarathy et al. (1992) Annu Rev Med 43:219-225). Foam cells, together with the binding of extracellular lipids to collagen fibers and proteoglycans, play an important role in the formation and growth of the lipid-rich atheroma.
Histopathologic examination of atheromatous plaques has revealed substantial variations in the thickness of fibrous caps, the size of the atheromas, the extent of dystrophic calcification and the relative contribution of major cell types (van der Wal et al. (1994) Coron Artery Dis 5:463-469). Resident cells present in active atheromatous plaques include a significant population of inflammatory cells, such as monocytes/macrophages and T lymphocytes. The emigration of monocytes into the arterial wall, and their subsequent differentiation into macrophages and ultimately foam cells, remains one of the earliest steps in plaque formation. Once there, these cells play a critical role in secreting substances that further contribute to atherosclerosis.
New therapies designed to target the genesis of atheromatous plaque and/or thrombus formation, or processes associated with atheromatous plaque and/or thrombus formation, as well as internal inflammation and infection are needed.
Current therapies designed to ameliorate the occlusive effects of atheromatous plaques on coronary blood flow, such as coronary artery bypass surgery and percutaneous transluminal coronary angioplasty, do not always prevent the incidence of acute coronary syndrome. Moreover, at least 50% of patients receiving angioplasty must return for a further procedure between 6 months to one year after the initial procedure. Acute coronary syndrome covers a group of sudden-onset coronary diseases, including unstable angina, acute myocardial infarction and sudden cardiac death. The causative agent of acute coronary syndrome is fissure, erosion or rupture of a specific kind of atheromatous plaque known as a “vulnerable plaque.” Vulnerable plaques are responsible for the majority of heart attacks, strokes, and cases of sudden death.
Post-mortem evidence suggests that vulnerable plaque rupture occurs in areas of the coronary arteries that are less than about 50% stenosed. Thus, angioplasty and bypass procedures, which are carried out on severely stenosed arteries, rarely remove vulnerable plaques or reduce the incidence of acute coronary syndrome (Plutzky (1999) Am J Cardiol 84:15J-20J). Even with currently available therapeutic approaches, such as lipid lowering, angioplasty and bypass, an unacceptably high incidence of acute coronary syndrome remains (Sacks et al. (2000) Circulation 102:1893-1900).
A vulnerable plaque is structurally and functionally distinguishable from a stable atheromatous plaque. For example, several histologic features distinguish a vulnerable plaque from a stable atheromatous plaque. A vulnerable plaque is characterized by an abundance of inflammatory cells (e.g., macrophages and/or T cells), a large lipid pool, and a thin fibrous cap.
Pathologic studies have provided a further understanding of why vulnerable plaques have a higher propensity for rupture than other atheromatous plaques. The thickness and integrity of the fibrous cap overlying the lipid-rich core is a principal factor in the stability of the plaque. Vulnerable plaques prone to rupture can be characterized as having thinner fibrous areas, increased numbers of inflammatory cells (e.g., macrophages and T cells), and a relative paucity of vascular smooth muscle cells. Vascular smooth muscle cells are the major source of extra cellular matrix production, and therefore, the absence of vascular smooth muscle cells from a vulnerable plaque contributes to the lack of density in its fibrous cap.
While the fibrous tissue within the cap provides structural integrity to the plaque, the interior of the atheroma is soft, weak and highly thrombogenic. It is rich in extracellular lipids and substantially devoid of living cells, but bordered by a rim of lipid-laden macrophages (van der Wal et al. (1999) Cardiovasc Res 41:334-344). The lipid core is a highly thrombogenic composition, rich in tissue factor, which is one of the most potent procoagulants known. The lesional macrophages and foam cells produce a variety of procoagulant substances, including tissue factor. The fibrous cap is the only barrier separating the circulation from the lipid core and its powerful coagulation system designed to generate thrombus. Essentially, the rapid release of procoagulants into the blood stream at the site of rupture forms an occlusive clot, inducing acute coronary syndrome. Thus, the thinner the fibrous cap, the greater the instability of the thrombogenic lipid core and the greater the propensity for rupture and thrombosis.
Several factors can contribute to the weakened state of the fibrous cap. In particular, inhibition of extracellular matrix production or degradation of extracellular matrix components adversely impacts the structural composition of the fibrous cap. Macrophages and T lymphocytes have been identified as the dominant cell types at the site of plaque rupture or superficial erosion, and each of these inflammatory cells contributes to the inhibitory and/or degradative pathways. Accelerated degradation of collagen and other matrix components is carried out by macrophage proteases, such as matrix metalloproteinases (“MMPs”), which are secreted at the site of the plaque. MMPs constitute an extensive family of enzymes, including interstitial collagenase (e.g., MMP-I), gelatinases (e.g., MMP-2, MMP-9), and stromelysin (e.g., MMP-3). Stromelysins can activate other members of the MMP family, causing degradation among many matrix constituents. The presence of T cells in the plaque can further contribute to weakening of the fibrous cap. Activated T cells produce and secrete interferon-γ, a potent inhibitor of collagen synthesis. Thus, the T lymphocytes represent a potentially large source of interferon-γthat can negatively regulate matrix production. Plaque rupture sites are further characterized by expression of major histocompatibility complex genes, (e.g., human lymphocyte antigen-DR on inflammatory cells and adjacent smooth muscle cells), indicating an active inflammatory reaction that also weakens the fibrous cap.
Present methods of plaque detection, several of which are discussed herein, are inadequate for detecting the genesis of atheromatous plaque and/or thrombus formation, or processes associated with atheromatous plaque and/or thrombus formation, as well as internal inflammation and infection. Present methods of plaque detection are also inadequate for detecting vulnerable plaques.
Common methods of plaque detection include angiography and angioscopy. Except in rare circumstances, angiography gives almost no information about the characteristics of plaque components. Angiography is only sensitive enough to detect hemodynamically significant lesions (>70% stenosis), which account for approximately 33% of acute coronary syndrome cases. Angioscopy is a technique based on fiber-optic transmission of visible light that provides a small field of view with relatively low resolution for visualization of interior surfaces of plaque and thrombus. Because angioscopic visualization is limited to the surface of the plaque, it is insufficient for use in detecting actively forming atheromatous and/or vulnerable plaques.
Several methods are being investigated for their ability to identify atheromatous plaques. However, none has proven to be sufficiently sensitive to identify vulnerable plaques or monitor the formation thereof. One such method, intravascular ultrasound (“IVUS”) uses miniaturized crystals incorporated at catheter tips and provides real-time, cross-sectional and longitudinal, high-resolution images of the arterial wall with three-dimensional reconstruction capabilities. IVUS can detect thin caps and distinguish regions of intermediate density (e.g., intima that is rich in smooth muscle cells and fibrous tissue) from echolucent regions, but current technology does not determine which echolucent regions are composed of cholesterol pools rather than thrombosis, hemorrhage, or some combination thereof. Moreover, the spatial resolution (i.e., approximately 2 cm) does not distinguish the moderately thinned cap from the high risk cap (i.e., approximately 25-75 μm) and large dense calcium deposits produce acoustic echoes which “shadow” so that deeper plaque is not imaged.
Intravascular thermography is based on the premise that atheromatous plaques with dense macrophage infiltration give off more heat than non-inflamed plaque (Casscells et al. (1996) Lancet. 347:1447-1451). The temperature of the plaque is inversely correlated to cap thickness. However, thermography may not provide information about eroded but non-inflamed lesions, vulnerable or otherwise, having a propensity to rupture.
Optical coherence tomography (“OCT”) measures the intensity of reflected near-infrared light from tissue. It provides images with high resolution that is approximately 10 to 20 times higher than that of IVUS resolution. OCT is primarily used for assessment of atherosclerotic plaque morphology. However, long image acquisition time, high costs, limited penetration and a lack of physiologic data render this approach undesirable for detection of actively forming atheromatous and/or vulnerable plaques.
Raman spectroscopy utilizes Raman effect: a basic principle in photonic spectroscopy named after its inventor. Raman effect arises when an incident light excites molecules in a sample, which subsequently scatter the light. While most of this scattered light is at the same wavelength as the incident light, some is scattered at a different wavelength. This shift in the wavelength of the scattered light is called Raman shift. The amount of the wavelength shift and intensity depends on the size, shape, and strength of the molecule. Each molecule has its own distinct “fingerprint” Raman shift. Raman spectroscopy is a very sensitive technique and is capable of reporting an accurate measurement of chemical compounds. Conceivably, the ratio of lipid to proteins, such as collagen and elastin, might help detect vulnerable plaques with large lipid pools. However, it is unlikely that actively forming and/or vulnerable plaques will be reliably differentiated from stable plaques based solely on this ratio.
All of the existing technologies and methods used to date are structural and therefore may be unable to detect actively forming or vulnerable plaques. All vascular detection agents known in the art involve the use of external imaging devices, such as gamma or positron cameras. The usefulness of such agents is limited and will not accurately detect plaque or thrombus due to the background activity from the surrounding tissue. Although 3D imaging via PET and SPECT is presently in use, the small size of the arteries as compared to the scatter from the large surrounding tissues lowers the utility of these imaging modalities as well.
Radiation-based methods for detection of diseased tissue are known in the art (U.S. Pat. No. 4,995,396). The devices of U.S. Pat. No. 4,995,396 are not designed to identify vulnerable plaques and further, U.S. Pat. No. 4,995,396 does not disclose intra-arterial beta probes. Use of beta-sensitive probes for the detection of plaques has been reviewed (Daghighian et al. Med Phys. 21:153-7(1994); U.S. Pat. Nos. 5,008,546, 5,744,805, 5,932,879, 6,076,009 and 6,295,680). U.S. Pat. No. 5,744,805 relates to an ion-implanted silicon radiation detector located at the tip of a probe with a preamplifier contained within the body of the probe, connected to the detector as well as external electronics for signal handling. U.S. Pat. Nos. 5,744,805 and 5,932,879 provide radio-pharmaceuticals for detecting diseased tissue, such as a cancerous tumor, followed by the use of a probe with one or more ion-implanted silicon detectors at its tip to locate the radiolabeled diseased tissue; the detector is preferentially responsive to beta emissions. U.S. Pat. Nos. 6,076,009, 5,568,532 and 5,864,141 relate to further designs for probes containing scintillators and photomultiplier tubes connected thereto. However, these techniques lack the precision of selective targeting as first described herein.
Photodynamic therapy (“PDT”) employs photoactivatable compounds known as photosensitizers to selectively target and destroy cells. Therapy involves delivering visible light of the appropriate wavelength to excite the photosensitizer molecule to the excited singlet state. This excited state can then undergo intersystem crossing to the slightly lower energy triplet state, which can then react further by one or both of two pathways, known as Type I and Type II photoprocesses (Ochsner (1997) J Photochem Photobiol B 39:1-18). The Type I pathway involves electron transfer reactions from the photosensitizer triplet to produce radical ions which can then react with oxygen to produce cytotoxic species such as superoxide, hydroxyl and lipid derived radicals. The Type II pathway involves energy transfer from the photosensitizer triplet to ground state molecular oxygen (triplet) to produce the excited state singlet oxygen, which can then oxidize many biological molecules such as proteins, nucleic acids and lipids, and lead to cytotoxicity.
Photodynamic therapy (PDT) has recently gained regulatory approval in the United States for treatment of esophageal cancer and in other countries for several other types of cancers (Dougherty et al. (1998) J Natl Cancer Inst 90:889-905). Certain photosensitizers accumulate preferentially in malignant tissues (Hamblin & Newman (1994) J Photochem Photobiol B 23:38), creating the advantage of dual selectivity: not only is the photosensitizer ideally specific for the target tissue, but the light can also be accurately delivered to the target tissue, thereby limiting the area within which the toxic effects of the photosensitizer are released.
Photodynamic therapy has been applied in cardiovascular medicine for two broad indications: treatment of atherosclerosis (“photoangioplasty”) and inhibition of restenosis due to intimal hyperplasia after vascular interventions (Rockson et al. (2000) Circulation 102:591-596, U.S. Pat. Nos. 5,116,864, 5,298,018, 5,308,861, 5,422,362, 5,834,503 and 6,054,449). Hematoporphyrin derivative (“HpD”) was the first of a number of photosensitizers with demonstrable, selective accumulation within atheromatous plaques (Litvack et al. (1985) Am J Cardiol 56:667-671). Subsequent studies have underscored the affinity of porphyrin derivatives for atheromatous plaques in rabbits and miniswine. There is maximal photosensitizer accumulation within the arterial intimal surface layers, which is diminished in comparison to the arterial media. Both HpD and Photofrin, a more purified derivative of HpD, also display in vitro preferential uptake by human atheromatous plaques. However, there is generally a relative lack of selectivity of most photosensitizers for atheromatous plaques and more particularly for vulnerable plaques. Moreover, methods known in the art for photodynamic destruction of atherosclerotic plaques generally fail as a result of the inflammatory response that follows PDT.
Recently, interventional strategies leading to vulnerable plaque stabilization have become an active area of research (Rabbani & Topol (1999) Cardiovasc Res 41:402-417). A therapy designed to detect, stabilize and reduce or eliminate active atheromatous and/or vulnerable plaques without inducing an inflammatory response would be highly desirable.